Abstract 6317: Targeting cell-surface CEA with a novel human fusion construct delivers the serine protease granzyme B to solid tumor cells
Authors: Khalid A. Mohamedali, Ana Alvarez-Cienfuegos, Lawrence H. Cheung, Walter N. Hittelman, Michael G. Rosenblum
Journal: Cancer Research (2023)

Abstract Cancer treatment has improved in the past decades due to advances in targeted therapies and surgical techniques. Immunotherapy, for example, has greatly improved outcomes for people with several types of cancer that typically have a poor prognosis. However, the response rate depends on the type of cancer being treated, with an average response rate between 20 to 50%. Thus, novel approaches to overcome such therapeutic challenges are warranted. Emerging therapies include fusion proteins and immunoconjugates that deliver a cytotoxic payload to the tumor cell with more specificity and potentially less avenues for resistance than exerted by the antibody or toxin alone. Carcinoembryonic antigen (CEA) is a cell surface protein normally found at low levels in adult tissues but over-expressed in a high percentage of solid tumors including colon and rectum, lung, prostate, ovary, thyroid, or liver. Thus, oncologic therapies against CEA may promote anti-tumor activity while sparing off-target toxicity to unaffected normal tissue. Our newly developed construct, designated GrB-Fc-huMFE, is a completely human fusion protein that contains the single-chain variable fragment huMFE antibody recognizing a juxta-membrane epitope of CEA remaining after proteolytic cleavage of CEA from the cell surface. The construct rapidly internalized into target cells resulting in granzyme B (GrB)-mediated mitochondrial depolarization and irreversible activation of both apoptotic and necrotic cascade mechanisms of cell death. In vitro cytotoxicity of GrB-Fc-huMFE against an initial subset of CEA+ tumor cell lines demonstrated cytotoxicity in the nanomolar range, compared to micromolar IC50 cytotoxicity against CEA-negative cell lines. Significantly, GrB-Fc-huMFE cytotoxicity in vitro was unaffected by exogenously-added CEA extracellular domain at doses up to 1000 μg/ml, suggesting that the construct is not competitively absorbed by soluble CEA proteolytically released by tumors and present in the circulation of patients. An in vivo efficacy study against established CEA+ (A-549) tumors in nude mice resulted in significant growth inhibition compared to vehicle controls (1200 mm3 vs &amp;lt;40 mm3). Complete tumor regression was observed in 7/10 tumors in mice treated (IP) with doses of 100 mg/kg of GrB-Fc-huMFE in a Q3Dx5 dose administration schedule. Mouse weight was unaffected in both treated and control groups for the duration of the study, suggesting no apparent toxicity of the construct. Pharmacokinetic, stability and in vitro tests against a broader panel of tumor cell lines are ongoing and will be reported. Altogether, GrB-Fc-huMFE appears to have significant potential in targeting a large category of CEA-expressing solid tumors and is a promising candidate for further pre-clinical and eventual clinical development. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Ana Alvarez-Cienfuegos, Lawrence H. Cheung, Walter N. Hittelman, Michael G. Rosenblum. Targeting cell-surface CEA with a novel human fusion construct delivers the serine protease granzyme B to solid tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6317.